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Keywords:

  • acetylcholinesterase;
  • Bombyx mori;
  • expression;
  • Km;
  • quantitative real-time polymerase chain reaction (qRT-PCR)

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Abstract  Two acetylcholinesterase (ace) genes have been reported in many insect species. In pests such as Helicoverpa assulta and Plutella xylostellas, ace1 gene encodes the predominant synaptic enzyme that is the main target of organophosphorus (OP) and carbamate pesticides. It has been reported that pesticide selection has an impact on the ace gene evolution. The domesticated silkworm, Bombyx mori, also has two ace genes. We studied ace gene expression and enzyme activities in silkworm as this has not faced pesticide selection over the past decades. The expression levels of two ace genes, Bm-ace1 and Bm-ace2, were estimated by quantitative real-time polymerase chain reaction. Bm-ace2 was expressed more highly than Bm-ace1 in all tested samples of different developmental stages or tissues, suggesting ace2, rather than ace1, is the major type of acetylcholinesterase (AChE) in Bombyx mori. This is inconsistent with the aforementioned lepidopterons agricultural pests, partly be due to the widespread use of pesticides that may induce high expression of the ace1 gene in these pests. Besides high expression in the head, Bm-ace1 also expresses highly in the silk glands and Bm-ace2 is abundant in the germline, implying both ace genes may have potential non-hydrolytic roles in development. Furthermore, we found that the mRNA levels of two ace genes and their ratios (ace2/ace1) change day to day in the first and third instars. This challenges the conventional method of estimating enzymatic activity using crude extract as an enzyme solution, as it is a mixture of AChE1 and AChE2. An efficient and simple method for separating different AChEs is necessary for reliable toxicological analyses.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Acetylcholinesterase (AChE, EC 3.1.1.7) is responsible for the hydrolysis of the neurotransmitter acetylcholine to terminate synaptic transmission (Toutant, 1989). In insects, there are two acetylcholinesterase (ace) genes, which were first independently reported in Anopheles gambiae (Weill et al., 2002) and Aphis gossypii (Li & Han, 2002). Since then, two ace genes have been found in many insect species, such as Culex pipiens (Huchard et al., 2006), Aedes aegypti (Mori et al., 2007) and Culex tritaeniorhynchus (Nabeshima et al., 2004), Myzus persicae (Nabeshima et al., 2003), Rhopalosiphum padi and Sitobion avenae (Chen & Han, 2006; Chen et al., 2007), Helicoverpa assulta (Lee et al., 2006), Plutella xylostella (Baek et al., 2005; Lee et al., 2007) and Bombyx mori (Seino et al., 2007). It was hypothesized that the two ace genes were derived from an old duplication before the diversification of the arthropods. An exception was observed in Drosophila melanogaster and other insects in the Cyclorrapha suborder of Dipterans. Only ace2 gene was found in these insects, possibly due to a second loss (Huchard et al., 2006; Weill et al., 2002).

The existence of two ace genes in insects has attracted increasing attention in the study of evolution and function of insect AChEs, especially their roles in conferring insecticide resistance. Previous studies showed that AChE1 is the principal target of organophosphorus (OP) and carbamate insecticides, indicating that ace1 gene encodes the main synaptic AChE in many agricultural pests (Alout et al., 2007; Lee et al., 2007; Malcolm et al., 1998; Nabeshima et al., 2003; Toda et al., 2004; Weill et al., 2002). The function of ace2 gene in these pests remains unclear. Generally, it is believed that insect ace1 has more important roles than ace2 (Lee et al., 2006, 2007). However, in Drosophila melanogaster (Weill et al., 2002), Lucilia cuprina (Chen et al., 2001) and Musca domestica, it is mutations of ace2 gene that contribute to insecticide resistance, suggesting that ace2 might also be the target of insecticides (Temeyer & Chen, 2007; Walsh et al., 2001).

Over the decades, insecticides have been widely used in pest control. As the target of organophosphorus (OP) and carbamate insecticides, ace genes have faced continuous selection pressure. This inevitably has impact on ace gene evolution. For example, it was found that the ace1 locus had duplicated in the past 40 years due to insecticide selection in C. pipiens (Labbe et al., 2007).

Most studies focus on agricultural or medical pests; ace genes in non-pest insects such as honeybee and silkworm are less well understood. Here, we took advantage of the domestic silkworm, Bombyx mori, which has not faced insecticide selection over the past decades, to investigate ace gene expression pattern. The availability of the genome sequence makes B. mori an ideal model organism for uncovering the evolution, expression and function of insect genes. We found that ace2 might be the major type of enzyme in B. mori, which is different from agricultural and medical pests.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Insects and tissues

Silkworm strain 7091 was obtained from the Sericulture Research Institute, Chinese Academy of Agricultural Science (Zhenjiang, China). Insects were maintained on an artificial diet at 26 ± 1°C and a 16 : 8 h L : D cycle. The specimens used in experiments were collected within 12 h after molting or at the same time on each day in the first and third instars. Six types of tissues (brain, enteron, malpighian tubules, silk gland, ovary and testis) were obtained from the fifth instar larvae before pupation.

Total RNA isolation and cDNA synthesis

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the recommended procedures. The RNA from different tissues were treated with DNAse I and then purified with phenol/chloroform treatment. The first strand of the complementary DNA (cDNA) template was synthesized from total RNA using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega, Mannheim, Germany) and Oligo (dT18) as the anchor primer. In reverse transcriptase polymerase chain reaction (RT-PCR), 60 ng total RNAs from the samples of different stages were used to synthesize a cDNA template. For the samples of different tissues, 100 ng total RNAs were used. The cDNA samples were quantified with Biophotometer (Eppendorf, Hamburg, Germany). All experiments were repeated in triplicates.

Quantitative real-time PCR

The messenger RNA (mRNA) levels of ace genes were estimated by quantitative real-time PCR (qPCR) using ABI Prism 7000 (ABI, Foster City, CA, USA). The qPCR reactions were carried out with SYBR Premix Ex Taq™ (Takara, Bio Inc., Shiga, Japan) following the manufacturer's protocol. The primers for qPCR were designed with Primer Premier 5.0 and the sequences are: ace1-F, 5′-AAGATTGGGGCGATGAAA-3′; ace1-R, 5′-GGACGTGGTCGAGGTGTC-3′; ace2-F, 5′-TACTTTTGCTCGATCTTGGG-3′; and ace2-R, 5′-ATGAGACCGCTCTTTGTTTC-3′. The ace gene sequences were downloaded from the Genbank database (DQ186605, DQ115792). The housekeeping gene β-actin A3 (X04507) was used as an internal control for normalization (Yao et al., 2005). Primers were: Actin-F, 5′-GGATGTCCACGTCGCACTTCA-3′ and Actin-R, 5′-GCGCGGCTACTCGTTCACTACC-3′. The qPCR reactions were performed following standard procedures which consisted of 95°C for 10 sec for initiation followed by 40 cycles of 95°C for 5 sec and 56°C for 31 sec. The specificity of the PCR reactions was monitored with gel electrophoresis and melting curve analysis using the Sequence Detection System (SDS). The PCR products were sequenced to verify accuracy. Amplification efficiencies were determined by the method of template dilution. The relative copy numbers of ace genes were calculated according to the 2−ΔΔCt method (Pfaffl, 2001). The mRNA levels of ace genes in the different tissues were analyzed using the Data Processing System (DPS). The difference was considered significant if P-value was <0.05 in Student's t-test.

Enzyme activity

The enzyme activities of AChEs were measured as described by Han et al. (1998) with minor modifications. Ten to fifty larvae in the third instar were homogenized in a glass tissue grinder on ice with phosphate buffer solution (0.02 mol/L, PH 7.0) containing 0.1% Triton X-100. The homogenate was centrifuged at 10 000 g for 20 min at 4°C and the supernatant was used as crude enzyme solution of AChE. The reaction mixture contained 50 μL phosphate buffer (0.02 mol/L, pH 7.0), 100 μL acetylthiocholine iodide (ATChI) (1.56 mmol/L), 100 μL dithiobisnitrobenzoic acid (DTNB) (45 μmol/L) and 50 μL enzyme solution. The reactions were measured at 405 nm using a Bio-Rad microplate reader (Bio-Rad, Hercules, CA, USA). The Km assay reactions contained in 300 μL : 50 μL phosphate buffer (0.02 mol/L, pH 7.0), 100 μL ATChI (12.5–1000 μmol/L), 100 μL DTNB (270 μmol/L) and 50 μL enzyme solution. All enzyme assays were repeated in triplicates.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

High expression of ace2 gene in the silkworm

Both ace genes were expressed in different stages including egg, the first to the fifth instar, pupa and adult. The mRNA levels were estimated with relative qPCR. We found that Bm-ace2 is more abundant than Bm-ace1 in all developmental stages. During egg, larvae and pupa stages, the ratio of ace2/ace1 ranges from 2.92 to 8.08. It increased to 25.54- (female) and 64.75-fold (male) at adult stage (Fig. 1). Interestingly, ace1 expresses more highly than ace2 in pests such as H. assulta (Lee et al., 2006) and P. xylostella (Baek et al., 2005).

image

Figure 1. The ratios of Bm-ace2/ Bm-ace1 in different developmental stages including egg, larva 1st–5th (L1–L5), pupa female & male (P-♀ & P-♂), adult female or male (A-♀ & A-♂). Data were analyzed by the equation, inline image.

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In C. pipiens, two ace genes exhibit similar expression pattern in different development stages (Huchard et al., 2006). However, Bm-ace1 and Bm-ace2 are not similar in B. mori. Bm-ace1 is highly expressed in the first instar but not in the adult. On the contrary, Bm-ace2 expresses highly both in the first instar and at the adult stage. Relative to the mRNA level of Bm-ace1 at egg stage (Bm-ace1egg), Bm-ace1 in the first instar (Bm-ace11st) is 40.12-fold and Bm-ace1male decreases to 6.24 in adult males. For ace2, Bm-ace21st is 1 12.98-fold whereas Bm-ace2adult is 266.20-fold relative to Bm-ace1egg (Table 1).

Table 1.  Relative expression levels of two ace genes at different developmental stages.
StagesBm-ace1Bm-ace2
  1. Relative to mRNA levels of Bm-ace1 in egg (Bm-ace1egg). *Student’s t-test, P < 0.05. Expression levels followed by the same letter showed no statistically significant difference.

Egg   1 b * 4.4 b
L140.12 a112.98 b
L211.00 b 29.72 b
L315.32 b100.31 b
L4 8.19 b 45.72 b
L5 8.41 b 72.85 b
P-♀ 9.44 b 47.02 b
P-♂ 6.37 b 34.00 b
A-♀ 3.59 b 71.55 b
A-♂ 6.24 b266.20 a

We noticed that β-actin A3 (X04507) might not be a good internal control as it does not expresses stably in different tissues (Mange et al., 1997; Mounier et al., 1991); the data were also analyzed without normalization using β-actin A3 as the reference, but relative to amount of total RNA in RT-PCR for synthesizing cDNA template. Generally, the results were similar with minor difference. Therefore, we have only provided the results analyzing with the 2−ΔΔCt method.

Daily changes of the Bm-ace2/Bm-ace1 ratio in the third instars

We examined the daily change of the two ace genes in the third instar because this stage is commonly used in insect toxicological experiments. Unexpectedly, the expression of two ace genes changes dynamically every day. The highest mRNA level of Bm-ace1 appears on day 3. The abundance of Bm-ace2 increases from day 1 to day 3 and rapidly decreases in the last two days (Fig. 2A). For each day, Bm-ace2 is more abundant than Bm-ace1. The ratio of Bm-ace2/Bm-ace1 also changes day-to-day and the highest ratio was 41.97 on day 2 (Fig. 2B).

image

Figure 2. Dynamic changes of ace1 and ace2 messenger RNA (mRNA) expression levels at each day in the third instar. (A) Relationship between enzyme activities and mRNA levels of two ace genes in the third instar. (B) The ratios of Bm-ace2/Bm-ace1 on each day in the third instar.

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Variation of enzymatic characteristics in the first and third instar

Crude extracts are often used as enzyme solution in insect toxicological experiments. We reasoned that enzyme characteristics might also be changeable every day because crude enzyme solution is the mixture of two AChEs. We used the crude extracts to measure AChE activities and found the enzymatic kinetic curves were dissimilar day-to-day in the first and the third instars (Fig. 3). In the third instar, enzyme activities were positively associated with the mRNA levels of Bm-ace2, suggesting that Bm-AChE2 is the predominant AChE in silkworms (Fig. 2A). Our work shows that dynamic expression of two ace genes leads to the alteration of AChE composition in the crude extract. Therefore, the results using crude extracts are not reliable.

image

Figure 3. The enzymatic kinetic curves of each day at the first instar (A) and at the third instar (B). The data on day 5 at the third instar were not provided because the enzyme activity was very low.

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Tissue-specific expression of two ace genes

Both ace genes in the silkworm exhibit tissue-specific expression patterns (Fig. 4A). Six types of tissues, head, enteron, malpighian tubules, silk gland, ovary and testis, were used for qRT-PCR analysis. As expected, both Bm-ace1 and Bm-ace2 were highly expressed in the head because AChE is a key enzyme in the nervous system. In addition, Bm-ace1 was also abundant in the silk gland, whereas Bm-ace2 was highly expressed in the ovary and testis, suggesting their potential roles in silk spinning or reproduction (Table 2). Consistent with the above results, Bm-ace2 was more abundant than Bm-ace1 in all tested tissues. The highest ratios were 46.01-fold in the ovary and 70.58-fold in the testis (Fig. 4).

image

Figure 4. The ratios of Bm-ace2/Bm-ace1 in different tissues. He: head; En: enteron; Ma: malpighian tubules; Si: silk gland; Ov: ovary, Te: testes.

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Table 2.  Relative expression levels of two ace genes in different tissues.
TissuesBm-ace1Bm-ace2
  1. Relative to ace gene expression in Malpighian tubules, separately.

Malpighian tubules 11
Enteron 0.620.36
Silk gland13.910.83
Testis 0.973.54
Ovary 3.405.48
Head44.789.72

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Since the first discovery of two insect ace genes in 2002, much attention has been paid to the expression and function of multiple ace genes, especially their roles in conferring insecticide resistance. In some agricultural pests, ace1, the paralog of Drosophila ace, was reported to be the principal target of insecticides and the predominant synaptic enzyme. The ace1 gene expresses more highly than ace2 in these pests. In H. assulta, Ha-ace1 was more abundant than Ha-ace2 in all tested tissues (Lee et al., 2006). In P. xylostella, the mRNA levels of Px-ace1 were 13- to 250-fold higher than that of Px-ace2 depending on the tissue (Baek et al., 2005). However, our results showed that Bm-ace2 is more abundant than Bm-ace1 in all tested samples, suggesting that Bm-AChE2, rather than Bm-AChE1, is the major type of enzyme in the silkworm. This is also confirmed by the results that AChE activities were positively associated with Bm-ace2. Another strain of silkworm was selected in the experiments, which also confirm that ace2 gene is more highly expressed than ace1 (data not provided). Although the expression level of a gene does not absolutely reflect its functional importance, high expression of Bm-ace2 in silkworm still suggests that ace2 gene has some unknown important roles. Our work may also partially explain the ultra-sensitivity of silkworm to insecticides, as the major AChE, Bm-AChE2, was reported to be more sensitive to the inhibitors than Bm-AChE1 (Shang et al., 2007).

We argue that the difference of ace gene expression pattern between the silkworm and pests is ascribed to different selection pressures. Over the past 50 years, pesticides have been major weapons against agricultural and medical pests. AChE is the target of widely used OP and carbamate insecticides. Therefore, ace genes in pests have faced continual selection by insecticides. In contrast, the silkworm is domesticated and does not face insecticide selection. It has been proven that exposure to organophosphate or bacterial infection induced the over-expression of AChE-R variant in mammals (Evron et al., 2007), suggesting OP and carbamate chemicals have the ability to affect ace gene expression. It is likely that exposure to OP and carbamate insecticides in the past decades induced the high expression of ace1 in pests. Besides insecticides, harsh conditions such as high temperature and food shortage in the wild could also be important factors affecting the evolution of ace gene as it participates in the response to environmental stimuli. The domesticated silkworms do not suffer from severe competition for food, partners and oviposition sites. In all, two ace genes exhibit different expression patterns in the silkworm and pests, possibly due to insecticides or environmental selection.

Although AChE purification has already been suggested for biochemical studies (Lee et al., 2006), the crude extracts are still commonly used to measure Ki, Km and I50 in toxicological experiments due to convenience. Previously, it has been believed that the complex components such as carboxylesterases in crude extracts make it unsuitable for enzyme analysis. In this work, we proved that AChE composition changes day-to-day because of the alternation of two ace genes’ expression levels. This indicates that experimental results using crude extracts are influenced not only by the complex components but also the relative abundance of different AChEs. Moreover, if AChEs are purified from crude extract but not separated individually, purified AChEs are still not suitable for toxicological analysis as it is the mixture of two AChEs and the ratio changes dynamically. Thus, an efficient and simple method for separating different AChEs is necessary for reliable toxicological analysis.

It has been suggested that AChE might have non-hydrolytic roles. In this work, we found that Bm-ace1 is highly expressed in the silk gland and Bm-ace2 is abundant in the ovary or testis. The high expression of ace gene in these tissues is unlikely to be responsible only for synaptic functions. It is possible that Bm-ace1 contributes to metamorphosis and Bm-ace2 participates in reproductive development. If this were the case, silkworm AChEs might have non-hydrolytic roles during development as proposed by Cousin et al., which requires further confirmation (Cousin et al., 2005; Zimmerman & Soreq, 2006).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank Miss Ling-Ling Wang's assistance in preparing total RNA from different tissues. This work was supported in part by the program of New Century Excellent Talents (NCET), the Foundation for the Author of National Excellent Doctoral Dissertation of PR China (FANEDD) and International Foundation for Science, Sweden (C/3800-1) to FL.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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